Elend C, Schmeisser C, Hoebenreich H, Steele H L, Streit W R
Biozentrum Klein Flottbeck, Abteilung Mikrobiologie, University Hamburg, Ohnhorststrasse 18, 22609 Hamburg, Germany.
J Biotechnol. 2007 Jul 15;130(4):370-7. doi: 10.1016/j.jbiotec.2007.05.015. Epub 2007 May 24.
We report on the isolation and biochemical characterization of a novel, cold-active and metagenome-derived lipase with a high stereo-selectivity for pharmaceutically important substrates. The respective gene was isolated from a cosmid library derived from oil contaminated soil and designated lipCE. The deduced aa sequence indicates that the protein belongs to the lipase family l.3, with high similarity to Pseudomonas fluorescens lipases containing a C-terminal secretion signal for ABC dependent transport together with possible motifs for Ca(2+)-binding sites. The overexpressed protein revealed a molecular weight of 53.2kDa and was purified by refolding from inclusion bodies after expression in Escherichia coli. The optimum temperature of LipCE was determined to be 30 degrees C. However, the enzyme still displayed 28% residual activity at 0 degrees C and 16% at -5 degrees C. Calcium ions strongly increased activity and thermal stability of the protein. Further detailed biochemical characterization of the recombinant enzyme showed an optimum pH of 7 and that it retained activity in the presence of a range of metal ions and solvents. A detailed analysis of the enzyme's substrate spectrum with more than 34 different substrates indicated that the enzyme was able to hydrolyze a wide variety of substrates including the conversion of long chain fatty acid substrates with maximum activity for pNP-caprate (C(10)). Furthermore LipCE was able to hydrolyze stereo-selectively ibuprofen-pNP ester with a high preference for the (R) enantiomer of >91% ee and it demonstrated selectivity for esters of primary alcohols, whereas esters of secondary or tertiary alcohols were nearly not converted.
我们报道了一种新型的、冷活性且源自宏基因组的脂肪酶的分离及生化特性，该脂肪酶对药学上重要的底物具有高立体选择性。从受油污染土壤的粘粒文库中分离出相应基因，并命名为lipCE。推导的氨基酸序列表明该蛋白属于脂肪酶家族l.3，与荧光假单胞菌脂肪酶高度相似，含有用于ABC依赖性转运的C端分泌信号以及可能的钙结合位点基序。过表达的蛋白分子量为53.2kDa，在大肠杆菌中表达后从包涵体中复性纯化。确定LipCE的最适温度为30℃。然而，该酶在0℃时仍显示28%的残余活性，在-5℃时为16%。钙离子强烈提高了该蛋白的活性和热稳定性。对重组酶的进一步详细生化特性分析表明其最适pH为7，并且在一系列金属离子和溶剂存在下仍保持活性。对该酶的底物谱进行详细分析，使用了34种以上不同底物，结果表明该酶能够水解多种底物，包括长链脂肪酸底物的转化，对对硝基癸酸酯（C(10)）具有最大活性。此外，LipCE能够立体选择性地水解布洛芬对硝基苯酯，对(R)对映体的优先选择性大于91%ee，并且它对伯醇酯具有选择性，而仲醇或叔醇酯几乎不被转化。
